Recent studies have refined our understanding of the mechanisms underlying pancreatic islet development from endoderm-derived pancreatic epithelium. However, these studies have not yet clearly defined the molecular and cell surface phenotype of islet precursor cells, not have they completely resolved the precise lineage relationships between islet precursor cells and mature islet endocrine cells. Progress in identifying and isolating islet progenitor/stem cells has been constrained by several factors including absence of unique surface markers, and difficulty in obtaining sufficient numbers of cells from embryonic tissues. Embryonic stem (ES) cells provide a useful in vitro model to study islet cytodifferentiation, and as such, could provide a potentially unlimited source of islet stem cells that would be relatively straightforward to isolate and manipulate in culture. This proposal will test the hypothesis that islet progenitor cells can be identified among differentiating murine ES cell derivatives and prospectively isolated using a transgenic lineage- selection strategy for subsequent expansion in culture. The specific aims of this proposal are: 1) To fully characterize committed pancreatic islet precursor cells in murine ES cell differentiation cultures; 2) To select and isolate PDX1+, neurogenin 3+, Nkx6.1+ pancreatic islet progenitor cells using a transgenic lineage selection strategy based on transgenic and "knock in" ES cells and retroviral transduction of ES cells; and 3) To identify culture conditions which allow their expansion and/or differentiation in vitro, and to measure their ability to reverse diabetes. A combination of indirect immunofluorescence and in situ hybridization will be used to characterize the pattern of pancreatic islet transcription factor and hormone expression. After deriving novel ES cell lines from transgenic and "knock in" mice, the generation of monoclonal antibodies to novel, unique cell surface markers and to identify novel markers through cDNA microarray expression profiling. Establishing pure cultures of large numbers of islet progenitor/stem cells through cDNA microarray expression profiling. Establishing pure cultures of large numbers of islet progenitor/stem cells from ES cells would have important implications for studying islet development and for providing a limitless source of islet cells for transplantation.